MUC1 is involved in trophoblast transendothelial migration

The factors that regulate trophoblast invasion of the uterine vasculature are incompletely understood. In this paper we show that macaque trophoblasts express the mucin, MUC1, and that it is involved in trophoblast-endothelial interaction. Immunocytochemistry, Western blotting and RT-PCR analyses confirmed that MUC1 was expressed by isolated early gestation macaque trophoblasts. MUC1 was also detected in endovascular trophoblasts in sections of placental-decidual tissue during early gestation. A blocking antibody against MUC1 reduced trophoblast adhesion to uterine endothelial cells and also blocked trophoblast transendothelial migration. MUC1 is known to bind to Intercellular Adhesion Molecule-1 (ICAM-1) in other systems. Incubation in the presence of a blocking antibody against Intercellular Adhesion Molecule-1 (ICAM-1) or recombinant ICAM-1 modestly, but significantly, reduced transendothelial trophoblast migration. These results are consistent with the idea that MUC1 is involved in trophoblast adhesion to uterine endothelial cells and in trophoblast transendothelial migration.     

MUC16 is produced in tracheal surface epithelium and submucosal glands and is present in secretions from normal human airway and cultured bronchial epithelial cells

The gel-forming MUC5AC and MUC5B mucins have been identified as major components of human airway mucus but it is not known whether additional mucin species, possibly with other functions, are also present. MUC16 mucin is a well-known serum marker for ovarian cancer, but the molecule has also been found on the ocular surface and in cervical secretions suggesting that it may play a role on the normal mucosal surface. In this investigation, the LUM16-2 antiserum (raised against a sequence in the N-terminal repeat domain) recognized MUC16 in goblet and submucosal gland mucous cells as well as on the epithelial surface of human tracheal tissue suggesting that the mucin originates from secretory cells. MUC16 mucin was present in 'normal' respiratory tract mucus as well as in secretions from normal human bronchial epithelial (NHBE) cells. MUC16 from NHBE cells was a high-molecular-mass, monomeric mucin which gave rise to large glycopeptides after proteolysis. N- and C-terminal fragments of the molecule were separated on gel electrophoresis showing that the MUC16 apoprotein undergoes a cleavage between these domains, possibly in the SEA domain as demonstrated for other transmembrane mucins; MUC1 and MUC3. After metabolic labeling of NHBE cells, most of the secreted monomeric, high-molecular-mass [(35)S]sulphate-labelled molecules were immunoprecipitated with the OC125 antibody indicating that MUC16 is the major [(35)S]sulphate-labelled mucin in NHBE cell secretions.     

MUC1 Limits Helicobacter pylori Infection both by Steric Hindrance and by Acting as a Releasable Decoy

The bacterium Helicobacter pylori can cause peptic ulcer disease, gastric adenocarcinoma and MALT lymphoma. The cell-surface mucin MUC1 is a large glycoprotein which is highly expressed on the mucosal surface and limits the density of H. pylori in a murine infection model. We now demonstrate that by using the BabA and SabA adhesins, H. pylori bind MUC1 isolated from human gastric cells and MUC1 shed into gastric juice. Both H. pylori carrying these adhesins, and beads coated with MUC1 antibodies, induced shedding of MUC1 from MKN7 human gastric epithelial cells, and shed MUC1 was found bound to H. pylori. Shedding of MUC1 from non-infected cells was not mediated by the known MUC1 sheddases ADAM17 and MMP-14. However, knockdown of MMP-14 partially affected MUC1 release early in infection, whereas ADAM17 had no effect. Thus, it is likely that shedding is mediated both by proteases and by disassociation of the non-covalent interaction between the α- and β-subunits. H. pylori bound more readily to MUC1 depleted cells even when the bacteria lacked the BabA and SabA adhesins, showing that MUC1 inhibits attachment even when bacteria cannot bind to the mucin. Bacteria lacking both the BabA and SabA adhesins caused less apoptosis in MKN7 cells than wild-type bacteria, having a greater effect than deletion of the CagA pathogenicity gene. Deficiency of MUC1/Muc1 resulted in increased epithelial cell apoptosis, both in MKN7 cells in vitro, and in H. pylori infected mice. Thus, MUC1 protects the epithelium from non-MUC1 binding bacteria by inhibiting adhesion to the cell surface by steric hindrance, and from MUC1-binding bacteria by acting as a releasable decoy.     

MUC1 mediates Pneumocystis murina binding to airway epithelial cells

Previous studies have shown that Pneumocystis binds to pneumocytes, but the proteins responsible for binding have not been well defined. Mucins are the major glycoproteins present in mucus, which serves as the first line of defence during airway infection. MUC1 is the best characterised membrane‐tethered mucin and is expressed on the surface of most airway epithelial cells. Although by electron microscopy Pneumocystis primarily binds to type I pneumocytes, it can also bind to type II pneumocytes. We hypothesized that Pneumocystis organisms can bind to MUC1 expressed by type II pneumocytes. Overexpression of MUC1 in human embryonic kidney HEK293 cells increased Pneumocystis binding, while knockdown of MUC1 expression by siRNA in A549 cells, a human adenocarcinoma‐derived alveolar type II epithelial cell line, decreased Pneumocystis binding. Immunofluorescence labelling indicated that MUC1 and Pneumocystis were co‐localised in infected mouse lung tissue. Incubation of A549 cells with Pneumocystis led to phosphorylation of ERK1/2 that increased with knockdown of MUC1 expression by siRNA. Pneumocystis caused increased IL‐6 and IL‐8 secretion by A549 cells, and knockdown of MUC1 further increased their secretion in A549 cells. Taken together, these results suggest that binding of Pneumocystis to MUC1 expressed by airway epithelial cells may facilitate establishment of productive infection.     

Tumor necrosis factor-alpha converting enzyme/ADAM 17 mediates MUC1 shedding

MUC1 clearance from the uterine epithelial cell surface is a prerequisite for the creation of an environment conducive to embryo implantation. In some species, reduced mRNA levels along with metabolic turnover account for loss of MUC1 during the receptive phase throughout the uterine epithelium. In other species, MUC1 is rapidly lost solely at the site of blastocyst attachment, suggesting the action of a protease. Correlative studies also indicate the presence of soluble forms of MUC1 in cell culture supernatants in vitro and in bodily fluids in vivo. To characterize the proteolytic activity mediating MUC1 release, shedding of MUC1 was analyzed in a human uterine epithelial cell line (HES) that abundantly expresses and readily sheds MUC1. MUC1 release was stimulated by phorbol 12-myristate 13-acetate and was markedly inhibited by the synthetic peptide hydroxamate metalloprotease inhibitor, tumor necrosis factor-alpha protease inhibitor (TAPI), as well as by an endogenous inhibitor of matrix metalloproteases, tissue inhibitor of metalloproteases (TIMP)-3. These characteristics along with studies conducted with cell lines genetically deficient in various ADAMs (for a disintegrin and metalloprotease) identified tumor necrosis factor-alpha converting enzyme (TACE)/ADAM 17 as a MUC1 sheddase. Furthermore, both TACE and MUC1 were expressed in human uterine epithelia during the receptive phase, and co-immunoprecipitation experiments revealed a physical interaction between TACE and MUC1 in HES cells. These studies establish a proteolytic mechanism for MUC1 clearance from a human uterine epithelial cell line and identify TACE as a MUC1 sheddase.     

MUC16 promotes EOC proliferation by regulating GLUT1 expression

As a common malignancy in females with a higher incidence rate, epithelial ovarian cancer (EOC) is a heterogeneous disease with complexity and diversity in histology and therapeutic response. Although great progress has been made in diagnosis and therapeutic strategies, novel therapeutic strategies are required to improve survival. Although the promoting effect of mucin 16 (MUC16) on tumour progression has been reported, the potential mechanisms remain unclear. In our study, we reported that overexpression of MUC16 was significantly related to cell proliferation and disease progression in EOC. Results from clinical specimen analysis and cell experiment support this conclusion. Patients with a high MUC16 expression usually had a worse prognosis that those with a low expression. Cell proliferation ability was significantly decreased in EOC cell lines when the knockdown of MUC16. Further study shows that the function of MUC16 in cell proliferation is based on the regulation of glucose transporter 1 (GLUT1) expression. MUC16 can control glucose uptake by regulating GLUT1 in EOC cells, thereby promoting glycogen synthesis, so that tumour cells produce more energy for proliferation. This conclusion is based on two findings. First, the significant correlation between MUC16 and GLUT1 was verified by clinical specimen and TCGA data analysis. Then, alteration of MUC16 expression levels can affect the expression of GLUT1 and glucose uptake was also verified. Finally, this conclusion is further verified in vivo by tumour-bearing mice model. To summarize, our results suggest that MUC16 promotes EOC proliferation and disease progression by regulating GLUT1 expression.     

Zonula occludens-1 and E-cadherin are coordinately expressed in the mouse uterus with the initiation of implantation and decidualization

Two-way interactions between the blastocyst trophectoderm and the uterine luminal epithelium are essential for implantation. The key events of this process are cell-cell contact of trophectoderm cells with uterine luminal epithelial cells, controlled invasion of trophoblast cells through the luminal epithelium and the basement membrane, transformation of uterine stromal cells surrounding the blastocyst into decidual cells, and protection of the "semiallogenic" embryo from the mother's immunological responses. Because cell-cell contact between the trophectoderm epithelium and the luminal epithelium is essential for implantation, we investigated the expression of zonula occludens-1 (ZO-1) and E-cadherin, two molecules associated with epithelial cell junctions, in the mouse uterus during the periimplantation period. Preimplantation uterine epithelial cells express both ZO-1 and E-cadherin. With the initiation and progression of implantation, ZO-1 and E-cadherin are expressed in stromal cells of the primary decidual zone (PDZ). As trophoblast invasion progresses, these two molecules are expressed in stroma in advance of the invading trophoblast cells. These results suggest that expression of these adherence and tight junctions molecules in the PDZ serves to function as a permeability barrier to regulate access of immunologically competent maternal cells and/or molecules to the embryo and provide homotypic guidance of trophoblast cells in the endometrium.     

Transmembrane and truncated (SEC) isoforms of MUC1 in the human endometrium and Fallopian tube

The cell surface mucin MUC1 is expressed by endometrial epithelial cells with increased abundance in the secretory phase of the menstrual cycle, when it is found both at the apical cell surface and in secretions. This suggests the presence of a maternal cell surface glycoprotein barrier to embryo implantation, arising from the anti-adhesive property of MUC1. In previous work, we demonstrated alternatively spliced MUC1 variant forms in tumour cells. The variant MUC1/SEC lacks the transmembrane and cytoplasmic sequences found in the full-length variant. We now show that MUC1/SEC mRNA is present in endometrial carcinoma cell lines, endometrial tissue and primary cultured endometrial epithelial cells. The protein can be detected using isoform-specific antibodies in uterine flushings, suggesting release from endometrium in vivo. However, on the basis of immunolocalisation studies, MUC1/SEC also remains associated with the apical epithelial surface both in tissue and in cultured cells. Transmembrane MUC1 and MUC1/SEC are both strikingly localised to the apical surface of tubal epithelium. Thus MUC1 may contribute to the anti-adhesive character of the tubal surface, inhibiting ectopic implantation. The mechanism by which this barrier is overcome in endometrium at implantation is the subject of ongoing investigation.     

Muc17 protects intestinal epithelial cells from enteroinvasive E. coli infection by promoting epithelial barrier integrity

The membrane-bound mucin MUC17 (mouse homolog Muc3) is highly expressed on the apical surface of intestinal epithelia and is thought to play a role in epithelial restitution and protection. Therefore, we hypothesized that MUC17 has a role in protection of the intestinal mucosa against luminal pathogens. Human intestinal cell lines were transfected by electroporation (Caco-2 and HT 29/19A) and by retroviral expression vector (LS174T, a cell line with high levels of MUC17 expression) using MUC17 siRNA. Transepithelial electrical resistance, permeability, tight-junction protein expression, adhesion, and invasion in response to enteroinvasive Escherichia coli (EIEC) were measured in all cell lines. In some experiments, the effect of the addition of exogenous purified crude mucin or recombinant Muc3 cysteine-rich domain protein (Muc3 CRD1-L-CRD2) as preventative or protective treatment was tested. Reduction of endogenous MUC17 is associated with increased permeability, inducible nitric oxide synthase and cyclooxygenase 2 induction, and enhanced bacterial invasion in response to EIEC exposure. Bacterial adhesion is not affected. Exogenous mucin (Muc3) and recombinant Muc3CRD treatment had a small but significant effect in attenuating the effects of EIEC infection. In conclusion, these data suggest that both native and exogenous MUC17 play a role in attachment and invasion of EIEC in colonic cell lines and in maintaining epithelial barrier function.     

Episialin (MUC1) overexpression inhibits integrin-mediated cell adhesion to extracellular matrix components

Episialin (MUC1) is a transmembrane molecule with a large mucin-like extracellular domain protruding high above the cell surface. The molecule is located at the apical side of most glandular epithelial cells, whereas in carcinoma cells it is often present at the entire surface and it is frequently expressed in abnormally large quantities. We have previously shown that overexpression of episialin reduces cell- cell interactions. Here we show that the integrin-mediated adhesion to extracellular matrix of transfectants of a melanoma cell line (A375), a transformed epithelial cell line (MDCK-ras-e) and a human breast epithelial cell line (HBL-100) is reduced by high levels of episialin. This reduction can be reversed by inducing high avidity of the beta 1 integrins by mAb TS2/16 (at least for beta 1-mediated adhesion). The adhesion can also be restored by redistribution of episialin on the cell surface by monoclonal antibodies into patches or caps. Similarly, capping of episialin on ZR-75-1 breast carcinoma cells, growing in suspension, caused adherence and spreading of these cells. We propose that there is a delicate balance between adhesion and anti-adhesion forces in episialin expressing cells, which can be shifted towards adhesion by strengthening the integrin-mediated adhesion, or towards anti-adhesion by increasing the level of expression of episialin.     

MUC16 in the lacrimal apparatus

The aim of the present study was to determine the possible expression of the mucin MUC16 in the lacrimal apparatus. Expression and distribution of MUC16 in lacrimal gland, accessory lacrimal glands, and nasolacrimal ducts was monitored by RT-PCR and immunohistochemistry. MUC16 was expressed and detected in all tissues investigated. Comparable to conjunctiva and cornea it was membrane-anchored in accessory lacrimal glands whereas in lacrimal gland acinar cells and columnar cells of the nasolacrimal ducts it was stored in intracytoplasmic vesicles without membrane-association. Subepithelial serous glands of the nasolacrimal ducts revealed staining of the secretion product. Intracelluar production of MUC16 is present in lacrimal gland and epithelial cells of the nasolacrimal ducts but it is not clear whether this MUC16 is secreted. MUC16 seems to be shedded or secreted from the epithelial surface of subepithelial serous glands of the nasolacrimal ducts. Our results show that MUC16 is present in the whole lacrimal apparatus. Its distribution pattern suggests different physiological functions with regard to tear film physiology and tear outflow. Moreover, the results demonstrate the existence of so far not recognized qualitative differences in the secretion product of main lacrimal gland and accessory lacrimal glands (glands of Krause).     

Expression of MUC1 mucin in human umbilical vein endothelial cells (HUVEC)

Mucin 1 (MUC1) is a membrane-bound glycoprotein that is expressed by various epithelial cell types. MUC1 functions include modulation of cell adhesion, signal transduction, lubrication and hydration of epithelial surfaces, and their protection from infection. In this study we demonstrated that MUC1 is expressed in human umbilical vein endothelial cells (HUVECs) and could be released/shed from cellular membrane. MUC1 presence in these cells was verified using three methods: Western blotting, flow cytometry and metabolic labeling. We also showed that mucin expression is stimulated by proinflammatory cytokines: about a 2-fold increase was observed after TNF-α treatment and lower after IFN-γ alone and in combination with TNF-α treatment. It can be assumed that the presence of MUC1 in endothelial cells may have an important role in the interactions with different cell types in physiological and pathological processes.     

MUC1 induced by Epstein-Barr virus latent membrane protein 1 causes dissociation of the cell-matrix interaction and cellular invasiveness via STAT signaling

Disruption of cellular adhesion is an essential pathobiologic step leading to tumor dissemination. Mucin 1 (MUC1) is a mucinous glycoprotein expressed at the surfaces of epithelial cells in many tissues and their carcinomas. MUC1 plays crucial roles in tumor invasion and metastasis, especially in opposing cell adhesion. We have shown that virus infection, specifically by the human tumor virus Epstein-Barr virus (EBV) induces a spectrum of cellular invasiveness and metastasis factors. Here we show that expression of MUC1 is increased in diverse latently EBV-infected cell lines that express latent membrane protein 1 (LMP1), the main viral oncoprotein, and that the level of MUC1 was suppressed by expression of a dominant-negative mutant of LMP1. Expression of LMP1 in EBV-negative nasopharyngeal cell lines induces expression of MUC1 through activation of the MUC1 promoter via binding of STAT1 and STAT3. Finally, LMP1 reduces cell adhesion ability, which is restored by inhibition of MUC1 expression with MUC1 small interfering RNA (siRNA). In addition, LMP1 increases cell invasiveness, which is suppressed by MUC1 siRNA. Thus, LMP1 induces MUC1, a factor important in an early step of detachment and release of tumor cells, which along with induction of other invasiveness and angiogenic factors may combine to act in a complex sequential process that culminates in metastasis of EBV-infected tumor cells.     

E-cadherin promotes EGFR-mediated cell differentiation and MUC5AC mucin expression in cultured human airway epithelial cells

In previous work, we showed that epidermal growth factor receptor (EGFR) activation causes mucin expression in airway epithelium in vivo and in human NCI-H292 airway epithelial cells and normal human bronchial epithelial (NHBE) cells in vitro. Here we show that the cell surface adhesion molecule, E-cadherin, promotes EGFR-mediated mucin production in NCI-H292 cells in a cell density- and cell cycle-dependent fashion. The addition of the EGFR ligand, transforming growth factor (TGF)-alpha, increased MUC5AC protein expression markedly in dense, but not in sparse, cultures. MUC5AC-positive cells in dense cultures contained 2 N DNA content and did not incorporate bromodeoxyuridine, suggesting that they develop via cell differentiation and that a surface molecule involved in cell-cell contact is important for EGFR-mediated mucin production. In support of this hypothesis, in dense cultures of NCI-H292 cells and in NHBE cells at air-liquid interface, blockade of E-cadherin-mediated cell-cell contacts decreased EGFR-dependent mucin production. E-cadherin blockade also increased EGFR-dependent cell proliferation and TGF-alpha-induced EGFR tyrosine phosphorylation in dense cultures of NCI-H292 cells, suggesting that E-cadherin promotes EGFR-dependent mucin production and inhibits EGFR-dependent cell proliferation via modulation of EGFR phosphotyrosine levels. Furthermore, in dense cultures, E-cadherin blockade decreased the rate of EGFR tyrosine dephosphorylation, implicating an E-cadherin-dependent protein tyrosine phosphatase in EGFR dephosphorylation. Thus E-cadherin promotes EGFR-mediated cell differentiation and MUC5AC production, and our results suggest that this occurs via a pathway involving protein tyrosine phosphatase-dependent EGFR dephosphorylation.     

Nuclear association of the cytoplasmic tail of MUC1 and beta-catenin

MUC1, an integral membrane mucin associated with the metastatic phenotype, is overexpressed by most human carcinoma cells. The MUC1 cytoplasmic tail (CT) is postulated to function in morphogenetic signal transduction via interactions with Grb2/Sos, c-Src, and beta-catenin. We investigated intracellular trafficking of the MUC1 CT, using epitope-tagged constructs that were overexpressed in human pancreatic cancer cell lines S2-013 and Panc-1. The MUC1 CT was detected at the inner cell surface, in the cytosol, and in the nucleus of cells overexpressing MUC1. Fragments of the MUC1 CT were associated with beta-catenin in both cytoplasm and nuclei. Overexpression of MUC1 increased steady state levels of nuclear beta-catenin but decreased nuclear levels of plakoglobin (gamma-catenin). There was no detectable association between plakoglobin and the MUC1 CT. Coimmunoprecipitation experiments revealed that the cytoplasmic and nuclear association of MUC1 CT and beta-catenin was not affected by disruption of Ca2+-dependent intercellular cadherin interactions. These results demonstrate nuclear localization of fragments of MUC1 CT in association with beta-catenin and raise the possibility that overexpression of the MUC1 CT stabilizes beta-catenin and enhances levels of nuclear beta-catenin during disruption of cadherin-mediated cell-cell adhesion.     

Increased adhesiveness in cultured endometrial-derived cells is related to the absence of moesin expression

Human endometrial epithelial cells (EECs) are nonadhesive for embryos throughout most of the menstrual cycle. During the so-called implantation window, the apical plasma membrane of EECs acquire adhesive properties by undergoing a series of morphological and biochemical changes. The human endometrial-derived epithelial cell line, RL95-2, serves as an in vitro model for receptive uterine epithelium because of its high adhesiveness for trophoblast-derived cells. In contrast, the HEC-1-A cell line, which displays poor adhesive properties for trophoblast cells, is considered to be less receptive. The ezrin, radixin, and moesin protein family members, which are present underneath the apical plasma membrane, potentially act to link the cytoskeleton and membrane proteins. In the present study, we have further investigated the adhesive features in these two unrelated endometrial-derived cell lines using an established in vitro model for embryonic adhesion. We have also analyzed the protein pattern and mRNA expression of ezrin and moesin in RL95-2 cells versus HEC-1-A cells. The results demonstrate that RL95-2 cells were indeed more receptive (81% blastocyst adhesion) compared with HEC-1-A cells (46% blastocyst adhesion). An intermediate adhesion rate was found in primary EECs cultured on extracellular matrix gel, thus allowing a partial polarization of these cells (67% blastocyst adhesion). Furthermore, we found that moesin was absent from RL95-2 cells. In contrast, ezrin is expressed in both cell lines, yet it is reduced in adherent RL95-2 cells. Data are in agreement with the hypothesis that uterine receptivity requires down-regulation or absence of moesin, which is a less-polarized actin cytoskeleton.     

Claudin 7 is reduced in uterine epithelial cells during early pregnancy in the rat

The non-receptive uterine luminal epithelium forms an intact polarised epithelial barrier that is refractory to blastocyst invasion. During implantation, organised dismantling of this barrier leads to a receptive state promoting blastocyst attachment. Claudins are tight junction proteins that increase in the uterine epithelium at the time of implantation. Claudin 7 is a member of this family but demonstrates a basolateral localisation pattern that is distinct from other claudins. The present study investigated the localisation, abundance and hormonal regulation of claudin 7 to elucidate a role for the protein during implantation. The results showed that claudin 7 demonstrates a distinct basal and lateral localisation in the uterine luminal and glandular epithelium throughout early pregnancy. On day 1, claudin 7 is abundantly present in response to ovarian estrogen. At the time of implantation, claudin 7 decreases in abundance. This decrease is not dependent on blastocyst presence, as shown by results in pseudopregnant animals. We propose that claudin 7 mediates intercellular adhesions in the uterine epithelium and also may be responsible for stabilising adhesion proteins at the basolateral cell surface. Thus, claudin 7 may function under the maintenance of the uterine luminal epithelial barrier, in the non-receptive state preventing implantation from occurring.